Compositions of nitrates and methods of use thereof

ABSTRACT

Inorganic anions nitrate and nitrite influence metabolic rate and glucose homeostasis. Infusion of nitrite iv caused an acute drop in resting energy expenditure (oxygen consumption) and nitrate, when given perorally, caused a reduction in oxygen consumption during exercise and a depression of the increase in blood glucose observed after an oral glucose tolerance test. The doses of nitrate and nitrite did not cause any detectable change in methemoglobin levels of blood. Also, nitrate and nitrite did not alter lactate levels in blood. This discovery provides useful treatments to regulate the energy expenditure and glucose homeostasis of a mammal by administration of inorganic nitrite and/or nitrate.

This application is a continuation application of earlier U.S. Utilitypatent application to Jon Lundberg and Eddie Weitzberg, entitled “Use ofNitrites and Nitrates and Compositions Containing These,” applicationSer. No. 12/528,794, filed Jun. 17, 2013, now pending, which is anational stage application of PCT application No. PCT/SE08/50212, filedFeb. 26, 2008, which claims the benefit of the filing date of U.S.Provisional Patent Application 60/919,709 to Jon Lundberg and EddieWeitzberg, filed on Mar. 22, 2007, the disclosures of all of which beinghereby incorporated entirely herein by reference.

This application is also a continuation application of earlier U.S.Utility patent application to Jon Lundberg and Eddie Weitzberg, entitled“Performance Enhancing Compositions and Use Thereof,” U.S. patentapplication Ser. No. 14/830,937, filed Aug. 20, 2015, now pending, whichis a continuation application of U.S. patent application Ser. No.12/528,798, filed Aug. 26, 2009, now issued as U.S. Pat. No. 9,180,140,which was a national stage application of PCT application No. PCT/SE2008/050211, filed Feb. 26, 2008, which is a claims priority to SwedishPatent Application 0700520-0, which was filed Feb. 26, 2007 and SwedishPatent Application 0700729-0, which was filed Mar. 22, 2007, thedisclosures of all of which being hereby incorporated entirely herein byreference.

FIELD OF THE INVENTION

The present invention relates to the field of performance enhancingnutritional foods and food supplements, liquid and solid edible productssuch as sport drinks, energy drinks and energy bars. The presentinvention also relates to the field of medicine and pharmaceuticals, inparticular pharmaceuticals and therapeutic methods for loweringmetabolic rate, oxygen consumption and/or glucose homeostasis in a humanpatient or another mammal, based on the administration of nitratesand/or nitrites to said patient or mammal.

BACKGROUND OF THE INVENTION

NO is involved in control of cellular respiration through interactionwith enzymes of the mitochondrial respiratory chain (for review seeMONCADA, S, et al. Does nitric oxide modulate mitochondrial energygeneration and apoptosis?. Nat Rev Mol Cell Biol. 2002, vol. 3, no. 3,p. 214-20). The classical means by which NO production occurs is theL-arginine pathway, where NO is synthesized by specific enzymes, theNO-synthases. A fundamentally different alternative way of generating NOhas been described more recently (LUNDBERG, J O, et al. Intragastricnitric oxide production in humans: measurements in expelled air. Gut.1994, vol. 35, no. 11, p. 1543-6; BENJAMIN, N, et al. Stomach NOsynthesis. Nature. 7 Apr. 1994, vol. 368, no. 6471, p. 502; ZWEIER, J L,et al. Enzyme-independent formation of nitric oxide in biologicaltissues. Nat Med. 1995, vol. 1, no. 8, p. 804-9; and WEITZBERG, E, etal. Nonenzymatic nitric oxide production in humans. NO Biol. Chem. 1998,no. 2, p. 1-7). In this NOS-independent pathway the inorganic anionsnitrate (NO₃ ⁻) and nitrite (NO.sub.2-) are reduced in vivo to form NO.Dietary nitrate (found mainly in green leafy vegetables) (MCKNIGHT, G M.Chemical synthesis of nitric oxide in the stomach from dietary nitratein humans. Gut. 1997, no. 40, p. 211-214; and Weitzberg, 1998, supra) isabsorbed from the circulation by the salivary glands, secreted in salivaand partly converted to nitrite in the oral cavity by nitrate reducingbacteria. Swallowed nitrite can then enter the systemic circulation.Indeed, a recent study shows that ingestion of nitrate results in asustained increase in circulating nitrite levels (LUNDBERG, J O, et al.Inorganic nitrate is a possible source for systemic generation of nitricoxide. Free Rad Bio Med. 2004, vol. 37, no. 3, p. 395-400). Furtherreduction of nitrite into bioactive NO can occur spontaneously in acidicor reducing environments (Benjamin et al. 1994, supra, Lundberg et al.1994, supra) but is also greatly enhanced by various proteins andenzymes including deoxyhemoglobin in blood (COSBY, K, et al. Nitritereduction to nitric oxide by deoxyhemoglobin vasodilates the humancirculation. Nat Med. 2003, vol. 9, no. 12, p. 1498-505), deoxymyoglobin(SHIVA, S. et al. Deoxymyoglobin is a Nitrite Reductase That GeneratesNitric Oxide and Regulates Mitochondrial Respiration. Circ Res. 9 Feb.2007), xanthine oxidase (MILLAR, T M, et al. Xanthine oxidoreductasecatalyses the reduction of nitrates and nitrite to nitric oxide underhypoxic conditions. FEBS Lett. 8 May 1998, vol. 427, no. 2, p. 225-8)and possibly by enzymes of the mitochondrial respiratory chain (forreview see LUNDBERG, J O, et al. Nitrate, bacteria and human health. NatRev Microbiol. 2004, vol. 2, no. 7, p. 593-602; LUNDBERG, J O, et al. NOgeneration from nitrite and its role in vascular control. ArteriosclerThromb Vasc Biol. 2005, vol. 25, no. 5, p. 915-22; and GLADWIN, M T, etal. The emerging biology of the nitrite anion. Nat Chem. Biol. 2005,vol. 1, no. 6, p. 308-14). NOS-independent NO production seems tocomplement the endogenous NO production especially during ischemia andacidosis when oxygen availability is low and the NO synthases operatepoorly (Zweier et al. 1995, supra; Weitzberg et al, 1998, supra;DURANSKI, M R, et al. Cytoprotective effects of nitrite during in vivoischemia-reperfusion of the heart and liver. J Clin Invest. 2005, vol.115, no. 5, p. 1232-40; Lundberg et al, 2004, supra). Tissue acidosisand relative hypoxia is present also during physical exercise and inthis metabolic state, bioactivation of nitrite is likely enhanced.

Recent studies indicate that nitrate and nitrite can have significantbiological effects in the body and that these effects may be beneficial(LUNDBERG, Jon O., et al. Nitrate, becteria and human health. Nat RevMicrobiol. 2004, no. 2, p. 593-602). For example, the nitrite anion cancause vasodilatation at near physiological concentrations when tested invitro (MODIN, A., et al. Nitrite-derived nitric oxide: a possiblemediator of ‘acidic-metabolic’ vasodilation. Acta Physiol Scand. 2001,vol. 171, p. 9-16) or when infused intra-arterially to humans (COSBY,K., et al. Nitrite reduction to nitric oxide by deoxyhemoglobinvasodilates the human circulation. Nat Med. 2003, no. 9, p. 1498-505).Nitrate can be converted to nitrite in vivo in a process dependent oncommensal bacteria (SPIEGELHALDER, B., et al. Influence of dietarynitrate on nitrite content of human saliva: possible relevance to invivo formation of N-nitroso compounds. Food Cosmet Toxicol. 1976, no.14, p. 545-548). When nitrate is ingested it is rapidly absorbed intoblood and then accumulates in saliva. In the oral cavity bacteria reduceparts of the dietary nitrate to nitrite and nitrite can then enter thesystemic circulation. (LUNDBERG, Jon O., et al. Inorganic nitrate is apossible source for systemic generation of nitric oxide. Free Radic BiolMed. 2004, vol. 37, p. 395-400).

In vitro studies published in the 1990s show that NO is a modulator ofmitochondrial respiration via reversible inhibition of cytochrome coxidase (CARR, G J. et al. Nitric oxide formed by nitrite reductase ofParacoccus denitrificans is sufficiently stable to inhibit cytochromeoxidase activity and is reduced by its reductase under aerobicconditions. Biochim Biophys Acta. 15 May 1990, vol. 1017, no. 1, p.57-62.; BOLANOS, J P, et al. Nitric oxide-mediated inhibition of themitochondrial respiratory chain in cultured astrocytes. J. Neurochem.1994, vol. 63, no. 2, p. 910-6; BROWN, G C, et al. Nanomolarconcentrations of nitric oxide reversibly inhibit synaptosomalrespiration by competing with oxygen at cytochrome oxidase. FEBS Lett.19 Dec. 1994, vol. 356, no. 2-3, p. 295-8; CLEETER, M W, et al.Reversible inhibition of cytochrome c oxidase, the terminal enzyme ofthe mitochondrial respiratory chain, by nitric oxide. Implications forneurodegenerative diseases. FEBS Lett. 23 May 1994, vol. 345, no. 1, p.50-4; and SCHWEIZER, M, et al. Nitric oxide potently and reversiblydeenergizes mitochondria at low oxygen tension. Biochem Biophys ResComm. 1994, no. 204, p. 169-75). NO may also interact at other sites ofthe mitochondrial respiratory chain and in the Krebs cycle (for reviewsee Moncada, supra). While this important action of NO has been verywell characterised in cell cultures, less is known about itsphysiological relevance in vivo. To date the focus among researchers hasbeen on the cardiovascular effects of nitrite after its in vivoreduction to the vasodilator nitric oxide (NO) (COSBY et al. (supra);DURANSKI, M. R., et al. Cytoprotective effects of nitrite during in vivoischemia-reperfusion of the heart and liver. J Clin Invest. 2005, vol.115, p. 1232-1240; GLADWIN, M. T., et al. The emerging biology of thenitrite anion. Nat Chem Biol. 2005, no. 1, p. 308-14; LARSEN, F. J., etal. Effects of dietary nitrate on blood pressure in healthy volunteers.N Engl J Med. 2006, vol. 355, p. 2792-3). WO 2005/004884 A (USGOVERNMENT ET AL.) 2005-01-20 and WO 2005/007173 A (US GOVERNMENT ETAL.) 2005-01-27 describe a method to administer a nitrite saltspecifically to obtain vasodilatation in a subject. No effects oflow-dose nitrate/nitrite on energy expenditure or glucose homeostasis orthe effects of NO on cellular respiration during physical exercise havebeen described.

Physiological adaptation to exercise involves major cardiovascular andmetabolic changes. Oxygen consumption increases dramatically in theactive muscles with a parallel increase in muscle blood flow. In theseprocesses, the endogenous gas nitric oxide (NO) plays an importantregulatory role. NO increases blood flow to the muscles and modulatesmuscular contraction and glucose uptake (for review see STAMLER, J S. etal. Physiology of nitric oxide in skeletal muscle. Physiol Rev. 2001,vol. 81, no. 1, p. 209-37).

The available information on the role of NO in healthy subjects and inparticular in athletes during work or exercise is both insufficient andcontradictory. Shen and colleagues showed that administration ofNOS-inhibitors in vivo during submaximal exercise leads to increasedoxygen consumption in dogs (SHEN, W. et al. Role of NO in the regulationof oxygen consumption in conscious dogs. Circulation Res. 1999, no. 84,p. 840-5) and Lacerda and colleagues showed similar results in rats(LACERDA, A C R, et al. Evidence that brain nitric oxide inhibitionincreases metabolic cost of exercise, reducing running performance inrats. Neuroscience Letters. 2006, no. 393, p. 260-3). The majority ofstudies have been done using NOS-inhibitors while the effects ofadministering exogenous NO on exercise are largely unknown. In addition,studies in healthy humans are scarce.

Interestingly, the marketing of some currently available foodsupplements for athletes and bodybuilders refer to the vasodilatoryeffect of NO. One example is “NOX2” (Bodyonics, Ltd., USA), a productsaid to contain arginine alpha-ketoglutarate (A-AKG) andarginine-ketoisocaproate (A-KIC) and allegedly capable of boosting shortterm nitric oxide levels. Other products contain L-arginine, from whichNO is synthesized by the NOS enzymes, and the beneficial effects of NOare often referred to, however without offering more detailedexplanations.

The relation between peak work rate and resting levels of nitrate inplasma and urine from subjects with different levels of physical fitnesshas been studied (Jungersten et al., Both physical fitness and acuteexercise regulate nitric oxide formation in healthy humans. J ApplPhysiol 82:760-764, 1997). A positive relationship between physicalfitness and formation of NO at rest was found and it was hypothesisedthat this positive relationship helps to explain the beneficial effectsof physical exercise on cardiovascular health. In Jungersten's studynitrate was used solely as a marker of NO production and the authorsstate several times that nitrate is a stable and inert end product of NOand that it is biologically inactive.

Nitrate (NO₃ ⁻) and nitrite (NO₂) are generally viewed as unwantedresidues in the food chain with potentially harmful effects (JointFAO/WHO Expert Committee on Food Additives (JECFA). Safety Evaluation ofCertain Food Additives. WHO, 1970. ISBN 9241660503; TANNENBAUM, S. R.,et al. Nitrite in human saliva. Its possible relationship to nitrosamineformation. J cancer Ins. 1974, vol. 53, p. 79-84; BARTSCH, H., et al.Inhibitors of endogenous nitrosation: mechanisms and implications inhuman cancer prevention. Mutation Res. 1988, vol. 202, p. 307-324).Proposed harmful effects of these anions include promotion of gastriccancers and other malignancies and development of methemoglobinemia ininfants. Because of this the levels of nitrate/nitrite are strictlyregulated in food and drinking water.

SUMMARY OF THE INVENTION

The disclosure is directed to methods of increasing exercise endurancein a human subject. The method comprising administering to a humansubject prior to exercise a composition comprising an amount ofinorganic nitrate (NO₃ ⁻), wherein amount of inorganic nitrate providesa supplementary amount of the inorganic nitrate. In some aspects, theadministration of the composition delays the onset of fatigue duringexercise; reduces the subject's oxygen consumption during exercise;and/or increases the available energy to the muscle. In someimplementations, the initial administration of the composition is atleast three days prior to exercise. In some aspects, the composition isadministered to the subject once a day. In some implementations, thecomposition is administered orally.

In some embodiments, the supplementary amount of the inorganic nitratein the composition administers between 0.01 mmol nitrate ion and 10 mmolnitrate ion per kg body weight of the subject or between 0.1 mmolnitrate ion and 1 mmol nitrate ion per kg body weight of the subject.For example, in one implementation, the supplementary amount of theinorganic acid in the composition administers 0.1 mmol nitrate ion perkg body weight of the subject.

In some implementations, the composition further comprises a polyphenol.The polyphenol is provided from at least one natural source, for examplefrom apple, pear, grapes, lemon, orange, lime, peach, pomegranate,grapefruit, kiwi, ginger, pineapple, blackberries, black raspberries,blueberries, cranberries, red raspberries, cherries, bog wortleberry,lingonberries, black elderberry, black chokeberry, black currant,cloudberries, strawberries, carrots, chili, rhubarb, onions, cacaoproducts, green tea, black tea, nuts, Yerba mate, or coffee. In suchcompositions, at least 50% of the composition is the inorganic nitrate.

In other implementation, the composition consists of the inorganicnitrate and at least one additive. In some aspects, the inorganicnitrate is provided from a natural nitrate source, for example, juice ordried concentrate from at least one of spinach, lettuce, fennel,cabbage, Chinese cabbage, and beetroot. In other aspects, the inorganicnitrate is in the form of a salt of nitrate, for example, sodiumnitrate, potassium nitrate, calcium nitrate, zinc nitrate, argininenitrate, or nitrate ammonium. The additive can be a natural flavor, anartificial flavor, a sweetener, a flavor enhancer, a color additive, anemulsifier, a stabilizer, fat, or a preservative.

In some implementations, the composition administered has the form of aliquid, a paste, a bar, a cake, a powder, a granulate, an effervescenttablet, a chewing gum, a tablet, a capsule, a lozenge, a fast meltingtablet or wafer, a sublingual tablet, or a spray or is a functional foodproduct. The functional food product can be a sport drink, an energydrink, a sport bar, or an energy bar. In some embodiments where thecomposition is a functional food production, the composition furthercomprises non-pathogenic live bacteria and/or a probiotic yeast. In someaspects, the non-pathogenic live bacteria is a species of Lactobacilli,Bifidobacteria, Veillonella, Staphylococcus, Actinomyces, or Rothia. Forexample, the Lactobacilli species is selected from L. acidophilus, L.delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L.plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum, or L.reuteri. The Bifidobacteria species is selected from B. breve, B.bifidum, or B. lactis.

Further embodiments will become evident to the skilled person upon studyof the figures, description and examples, as well as the appendedclaims, incorporated herein by reference.

DESCRIPTION OF THE FIGURES

The invention will be described in closer detail in the followingdescription, examples and non-limiting claims, with reference to theattached drawings in which:

FIG. 1 shows a graph illustrating numerous ways in which the combinationof nitrate and polyphenols synergistically act to increase thebioavailability of nitric oxide and at the same time to reduce theformation of harmful compounds such as oxygen radicals and nitrosamines.For detailed explanation see text.

FIG. 2 is a graph showing changes in oxygen consumption (VO₂) followingiv infusion of sodium nitrite in increasing doses. Nitrite was infusedover a 10 min period in non-smoking healthy male volunteers (30-70years).

FIG. 3 is a graph showing the effects of a dietary supplementation withsodium nitrate or sodium chloride (placebo) on plasma concentrations ofnitrite measured at rest and immediately after exercise in 9 healthymale volunteers.

FIG. 4 is a bar diagram showing the oxygen consumption (VO₂) and heartrate (HR) measured at 6 different work rates after a 3-day dietarysupplementation with sodium nitrate (0.1 mmol/kg body weight/min, NIT)or an equal amount of sodium chloride (CON). The study had a randomizeddouble-blind cross-over design with a washout period of at least 10 daysbetween the tests. * p<0.05, ** p<0.01.

FIG. 5 is a graph showing oxygen consumption during bicycle exercise at80% of VO₂peak in 9 healthy male volunteers. Measurements were madeafter a 3-day dietary supplementation with sodium nitrate (0.1 mmol/kgbody weight/day) or an equal amount of sodium chloride (placebo). Thedifference between nitrate and placebo periods was significant (p<0.01).

FIG. 6 is a bar diagram showing plasma lactate concentration measured at6 different work rates after dietary supplementation with sodium nitrate(0.1 mmol/kg body weight/day for 3 days, filled bars) or an equal amountof sodium chloride (placebo, empty bars).

FIGS. 7a-7c consist of three graphs, showing changes in blood glucoselevels after an oral challenge with glucose for three test subjects in adouble-blind, placebo-controlled cross-over study (FIGS. 7a, 7b and 7c). A standard oral glucose tolerance test was performed. The subjects(healthy non-smoking volunteers) had their diet supplemented for 3 dayswith either sodium chloride (placebo) or sodium nitrate (Nitrate) at adose of 0.1 mmol/kg body weight/day.

FIG. 8 is a graph showing changes in blood glucose levels after an oralchallenge with glucose in 8 additional subjects in a double-blind,placebo-controlled cross-over study. A standard oral glucose tolerancetest was performed. The subjects (healthy non-smoking volunteers) hadtheir diet supplemented for 3 days with either sodium chloride (PLACEBO)or sodium nitrate (NITRATE) at a dose of 0.1 mmol/kg/day. Data arepresented as mean±SEM.

FIG. 9 is a graph showing the effect of a two-week intervention withbeetroot juice (fresh juice 3-4 dl/day) on systolic, diastolic and meanarterial (MAP) blood pressure in a 43-year-old male with hypertension.

FIG. 10 shows the plasma nitrate and nitrite concentrations afterintravenous infusion of nitrate. Panel a) shows plasma nitrateconcentration; panel b) shows plasma nitrite concentrations; and panelc) shows plasma nitrite concentrations in wild type (C57BL/6), germ freeand knockout (eNOS) mice.

FIG. 11 is a graph showing enhanced post-ischemic blood flow afternitrate infusion.

DETAILED DESCRIPTION

Before the present method and compositions are described in the form ofembodiments thereof, it is to be understood that this invention is notlimited to the particular configurations, method steps, and materialsdisclosed herein as such configurations, steps and materials may varysomewhat. It is also to be understood that the terminology employedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims and equivalentsthereof.

It must also be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

The term “about” when used in the context of numeric values denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. Said interval can be +/−2% of the given value, preferably +/−5%,and most preferably +/−10% of the numeric values, where applicable.

The term “indirect calorimetry” is here defined as a method forcalculating heat that living organisms produce from their production ofcarbon dioxide and nitrogen waste and from their consumption of oxygen,well known to persons skilled in the relevant art.

The term “catabolism” is defined as the metabolic process that breaksdown molecules into smaller units. It is made up of degradative chemicalreactions in the living cell.

The term “edible” in this context means non-toxic and possible toingest, however not limited to particular modes of ingesting, such asdrinking, chewing, applying to the oral cavity in various forms, suchas, for example a spray or aerosol.

The term “energy expenditure” is here defined as the amount of energyexpended for a certain metabolic rate.

The term “functional food” relates to any fresh or processed foodclaimed to have a health-promoting and/or disease-preventing propertybeyond the basic nutritional function of supplying nutrients. Functionalfoods are sometimes called nutraceuticals. The general category includesprocessed food made from functional food ingredients, or fortified withhealth-promoting additives, like “vitamin-enriched” products, and also,fresh foods (e.g. vegetables) that have specific claims attached.Fermented foods with live cultures are often also considered to befunctional foods with probiotic benefits.

The term “insulin resistance” is here defined as a condition in whichnormal amounts of insulin are inadequate to produce a normal insulinresponse from fat, muscle and liver cells.

The term “mammal” is intended to encompass all mammals, and inparticular humans, pets and agriculturally significant animals, as wellas animals used in competitions, such as horses and dogs.

The term “metabolic syndrome” is here defined as a combination ofmedical disorders that increase the risk for cardiovascular disease anddiabetes in a human. Symptoms and feature include fastinghyperglycaemia, diabetes mellitus type 2 or impaired fasting glucose,impaired glucose tolerance or insulin resistance, high blood pressure,central obesity, decreased HDL cholesterol, elevated triglycerides andelevated uric acid levels.

The term “metabolism” is used to define the complete set of chemicalreactions that occur in living cells and “metabolic rate” is defined asthe speed of metabolism of a mammal.

Methemoglobin is a form of hemoglobin in which the iron in the hemegroup is in the Fe₃ ⁺ state, not the Fe₂ ⁺ of normal hemoglobin.Methemoglobin is unable to carry oxygen. Methemoglobinemia is defined asa blood disorder characterized by the presence of a higher than normallevel of methemoglobin in the blood.

The term “oxygen consumption” is defined as the amount of oxygen (O₂)consumed by a mammal and is usually expressed as ml of pure oxygenconsumed/min. “Oxygen consumption” relates to the amount of oxygenconsumed by a mammal as whole but also to oxygen consumption in anisolated tissue or organ, such as, but not limited to, heart, liverbrain or other tissue exposed to ischemia.

The term “performance enhancing food or food supplement” includes sportdrinks and energy drinks, as well as other liquid, semi-solid or solidforms, such as energy bars and tablets. No distinction is intendedbetween sports drinks and energy drinks. Sports drinks tend to be moreisotonic while energy drinks tend to contain more sugar and frequentlyalso contain caffeine. Usually, sports drinks are non-carbonated andfrequently contain fructose or other sugars, and complex carbohydrates,which are easily absorbed by the body, and are designed to promote theavailability of energy and/or prevent or treat mild dehydration. Sportdrinks also contain electrolytes (mainly sodium and potassium salts) andnutrients (proteins and amino acids). Sport drinks, energy drinks andother liquid, semi-solid and solid products, while marketed forathletes, are also consumed by non-athletes, as a snack, in situationswhere extra energy and endurance is desired. It is currently believedthat improved sports performance can be attained by the intake ofso-called sport drinks.

The term “significant hypotension” means in this context an acutereduction of systolic and/or diastolic blood pressure, accompanied byclinical symptoms of hypotension such as dizziness, nausea, pallor, lossof consciousness, etc. Said symptoms may occur in various degrees, andit is preferred that they are entirely avoided, minimized or eliminatedas far as possible, or at least to an extent that they are clinicallyinsignificant.

The inventors have surprisingly shown that the metabolic rate and/or theoxygen consumption can be influenced in a mammal (locally orsystemically), by administering inorganic nitrite (NO₂ ⁻) and/or nitrate(NO₃ ⁻) to said mammal in an amount of nitrite and/or nitrate sufficientto decrease oxygen consumption. The decreased oxygen consumption isachieved without causing significant hypotension and without causing anysignificant increase of the methemoglobin level in said mammal. In caseof local reduction of the metabolic rate, the oxygen consumption isdecreased in an isolated tissue or organ such as the heart, liver, brainor other tissue that is exposed to ischemia (a condition in which bloodflow, and thus oxygen, is restricted to a part of the body). In suchcases the interaction of reaction products (including NO) of nitriteand/or nitrate with enzymes of the mitochondrial respiratory chain andsubsequent inhibition of respiration leads to lowering of oxygen demand,which is beneficial for an ischemic tissue. This effect resembleshibernation. Because the generation of active nitrite and/or nitratereaction products is maximized in ischemic tissues the effect of oxygenconsumption will be most pronounced at these sites. In one particularembodiment, the oxygen consumption is lowered in the heart.

The inventors also showed that dietary supplementation with inorganicnitrate results in a reduced VO₂ during physical exercise and asignificant increase in muscular efficiency. These effects occurredwithout any increase in plasma lactate.

In principle, also other additional approaches can lead to increasedsystemic or local nitrite levels. The most obvious is to administernitrite or its precursor nitrate as such, but this may be supplementedby or enhanced by increasing the gastric pH (e.g. with an acidsuppressive drug such as a proton pump inhibitor or H₂ receptorantagonist or antacids) to maximize nitrite survival in the stomach andthereby the systemic delivery of nitrite. Alternatively, theadministration of nitrite or nitrate can be supplemented by or enhancedby interfering with the oral microflora in order to maximize the numberof nitrate reducing species. This can be achieved through the deliveryof “probiotic” nitrate reducing bacteria or selective treatment with anantibiotic to favor the nitrate reducing species.

The surprising finding that nitrite and its precursor nitrate affectssuch vital physiological processes as metabolic rate and/or oxygenconsumption can be used therapeutically, e.g. in prophylaxis,alleviation or treatment of several conditions. In an attempt toincrease systemic nitrite levels, nitrite and/or nitrate can be given byenteral administration (orally, in the form of a liquid, semi-solid orsolid preparation, such as a chewing gum, tablet, lozenge, wafer, cake,bar or the like) or by parenteral administration (intravenous,transdermal, transcutaneous, by inhalation, rectally, vaginally,topical, intraperitoneally, intra muscular, subcutaneous, sublingual orany other way of parenteral administration). The nitrite and/or nitratecomprising composition and possible further combinations describedherein can be administered continuously or as single bolus doses. Insome aspects, the inventors make available a composition, preferably anedible composition, capable of enhancing performance manifested as areduced oxygen uptake (VO₂) during physical exercise when ingested by amammal, wherein said composition comprises inorganic nitrate and/ornitrite, and in particular a composition wherein the effect of enhancedperformance is manifested as both a reduced oxygen uptake (VO₂) duringphysical work and a significant increase in muscular efficiency.

Composition

It is likely that an optimal dose-interval exists, meaning that below acertain plasma level of nitrite the effects are insufficient and,correspondingly, that over a certain level the effect is lower andpossibly accompanied by side effects. In one embodiment, the compositioncomprising inorganic nitrite and/or nitrate is a pharmaceuticalcomposition comprising inorganic nitrite and/or nitrate in an amountwhich is sufficient to decrease oxygen consumption, but which does notincrease the methemoglobin level in a subject when administered to saidsubject in a prescribed dose. Optionally the composition comprisesanother pharmaceutically active compound.

According to a particular embodiment, said composition comprises nitritealone, without the presence of nitrate. According to another embodiment,said composition in addition to nitrate and/or nitrite, also comprisesarginine.

According to one embodiment, nitrate and nitrite are given in a doseratio interval of about 5:1 to about 100:1 (nitrate:nitrite), such as5:1, 10:1, 30:1, 50:1, 70:1 and 100:1. Preferably the dose ratio isabout 10:1. This will ensure acute effects of the nitrite as soon as itis absorbed, and then provide a sustained effect of the nitratefollowing its bioconversion into nitrite.

Preferably the dose of nitrate is about 1-1000 umol sodium nitrate/kgbodyweight/day. Correspondingly, the dose of nitrite is preferably about0.1-100 umol/kg bodyweight/day. More specifically, for the use of anitrate salt perorally, a dose of about 0.01-100 mmol/kg/24 h iscurrently preferred or more preferably a dose of about 0.01-10mmol/kg/24 h, even more preferably 0.1-1 mmol/kg/24 h. Correspondingly,the dose of nitrite is about 0.001-10 mmol/kg/24 h, preferably about0.001-1 mmol/kg/24 h and more preferably about 0.0001-0.1 mmol/kg/24 h.

Using intravenous administration, the nitrite is preferably administeredin a dose within the interval of about 0.01 to about 10000 nmoles/kgbody weight/min, preferably about 0.01 to about 1000 nmoles/kg bodyweight/min, more preferably about 0.1 to about 100 nmoles/kg bodyweight/min, most preferably about 1 to about 20 nmoles/kg bodyweight/min. It is presently contemplated that the most preferred dose isless than about 15 nmoles/kg body weight/min. For comparison, it shouldbe noted that the nitrite dose used in the treatment of cyanidepoisoning is about 100000 nmoles/kg body weight, or about 300-400 mggiven as a single dose. The nitrite can also be administered as one ormore bolus doses, preferably 0.01-100 umol/kg body weight, morepreferably 0.1-10 umol/kg body weight, and even more preferably 0.1-2umol/kg body weight.

For the use of a nitrate salt perorally, a dose of about 0.01-100mmol/kg body weight/24 h is currently preferred or more preferably adose of about 0.01-10 mmol/kg body weight/24 h, even more preferably0.1-1 mmol/kg body weight/24 h.

Importantly, the administered dose of nitrate or nitrite should notinduce production of more than about 10% methemoglobin, preferably notmore than about 5% methemoglobin, and more preferably not more thanabout 2% methemoglobin. Most preferred is that the dose of nitrate ornitrite does not induce any measurable change in methemoglobin in thesubject when administered or ingested according to the prescribed dose.

Pharmaceutically acceptable salts of nitrate and nitrite include but arenot limited to sodium, potassium, calcium, zinc, arginine and ammonium.Sodium and potassium salts are presently most preferred.

The nitrite and nitrate salts may be of synthetic origin, but may alsobe isolated from natural sources, such as naturally nitrate containingplants, e.g. green leafy vegetables, examples include, but are notlimited to spinach, lettuce, fennel, cabbage, Chinese cabbage andbeetroot. Concentrates, such as juices or dried concentrates of theseand other nitrate-rich vegetables or fruits are suitably used for themanufacture of food products (including functional food products) ornutritional supplements according to the present invention. In onepreferred embodiment, the nitrate in the inventive compositionoriginates from beetroot (such as beetroot juice). In yet anotherembodiment of the present invention a nitrate and/or nitrite salt (forexample potassium the natural nitrate source includes a dried vegetablepowder. In some aspects, the dried vegetable powder is used incombination with licorice, for example salty licorice (ammoniumchloride).

Alternatively, the composition comprising nitrite and/or nitrate is anutritional supplement, an enteral nutritional solution, an infantformula, a snack product or a parenteral nutritional solution comprisinginorganic nitrite and/or nitrate in an amount which is sufficient todecrease oxygen consumption, but which does not cause significanthypotension in a subject when ingested by said subject in a prescribeddose.

The inventive composition preferably has the form of a liquid, a paste,a bar, a cake, a powder, a granulate, an effervescent tablet, a chewinggum, a tablet, a capsule, a lozenge, a fast melting tablet or wafer, asublingual tablet or a spray. Another composition is a nicotine-freesmokeless tobacco and/or wet snuff. Such products can be manufacturedusing conventional methods practiced in the food and beverage industry,or in pharmaceutical industry. More preferably said composition is inthe form of, or constitutes a part of, a food product, such as a liquid,a paste, a bar, a cake, a powder, or a granulate.

According to a preferred embodiment, the composition according to theinvention is prepared as a fermented food product, such as a yogurt orsimilar dairy or non-dairy product, comprising a source of nitrateand/or nitrite in addition to live bacteria capable of enhancing nitrateor nitrite reduction.

According to another embodiment, the composition is presented to themarket as a functional food product.

The present inventors consider presenting the composition to the marketin the form of a sport drink, an energy drink, a sport bar, or an energybar. The energy bar may take on a variety of forms. For convenience, itis preferred that the energy food product be shaped like a box, square,cylinder, string, pie, sphere, triangle, or other format suitable forpackaging, transportation, handling and eating. For example, an energybar according to the present invention may include, in addition tonitrate and optionally nitrite, also a variety of other components suchas, for example, nuts, crisps, fruit pieces, chocolate, seeds, and thelike. Preferred nuts are almonds, peanuts, hazelnuts, cashews, walnuts,pecans, brazil nuts, and the like. Crisp components include rice crisps,corn crisps, oats, wheat flakes, and the like. The chocolate can be anytype of chocolate or chocolate like edible component in various forms,such as, for example, chocolate chips, chunks, flakes and the like.Non-limiting examples of seeds include sesame, sunflower, poppy,caraway, fennel and the like.

Products comprising the inventive composition can easily be manufacturedby persons skilled in the food, sweets and beverage industry or thepharmaceutical industry, and existing compositions supplemented withnitrate, nitrite and other combinations described herein in amountsaccording to this invention.

Additionally, traditional food ingredients such as flavors and the likemay be included. For example, additional ingredients may include naturaland artificial flavors, sweeteners, salt, flavor enhancers, coloradditives, emulsifiers, stabilizers, fats, preservatives, and the like.

In one embodiment of the present invention inorganic nitrite and/ornitrate is administered or used in combination with a polyphenol richcompound or product. Polyphenols are a group of chemical substancesfound in plants, characterized by the presence of more than one phenolgroup per molecule. Polyphenols are generally further subdivided intohydrolysable tannins, which are gallic acid esters of glucose and othersugars; and phenylpropanoids, such as lignins, flavonoids, and condensedtannins. In one embodiment of the present invention the inorganicnitrite and/or nitrate is/are mixed with a compound that contains highlevels of polyphenols. It is contemplated that this combination willhave synergistic health promoting effects via potentiation of NObioavailability. In some aspects, the combination of nitrite/nitrate andpolyphenol rich compound or product will act synergistically to enhanceNO formation in the body at the expense of detrimental compounds such asnitrosamines. The beneficial effects of this includes a reduction inblood pressure and platelet aggregation, reduced atherosclerosis,reduced risk of myocardial infarction, stroke and other cardiovasculardisorders, reduced risk of cancer in any form. Polyphenols will enhanceNO generation by several separate mechanisms highlighted in FIG. 1.First, such agents can directly stimulate endogenous NO formation fromNO synthase enzymes (1 in FIG. 1). Second, it is contemplated that thesecompounds will enhance the reduction of nitrite to bioactive NO due tothe presence of reductive —OH groups on the phenol ring (2 in FIG. 1).Third, by acting as scavengers of free radicals such as superoxide, theyprevent these radicals from interacting with (and destroying) NO andthereby, NO becomes more long-lived (3 in FIG. 1). In addition to this,nitrite or its reaction products can interact with the polyphenol itselfand modify it chemically via nitration or nitrosation reactions (4 a inFIG. 1). The resulting compound can act as a long-lived NO donor (4 b inFIG. 1). An additional effect is that the presence of polyphenols willdivert the chemical reactions away from formation of potentiallycarcinogenic nitrosamines (5 in FIG. 1). Nitrates reaction productnitrite can react with amines to form nitrosamines but polyphenols willinhibit this reaction by a dual mechanism. First, they help to rapidlyreduce HNO₂ directly to NO thereby minimizing the formation ofnitrosating species (N₂O₃, HNO₂). Second, they can directly compete fornitrosation with the amines by being nitrosated themselves.

The use of nitrite and/or nitrate or a combination of nitrate and/ornitrite withpolyphenols may also have beneficial effects on the bloodpressure. Thus, in one embodiment the present invention relates to amethod to reduce blood pressure, preferably to normal levels (about140/80 mmHg). Examples of nitrite and/or nitrate sources are naturalsources of nitrate (such as vegetables as mentioned above or juicesthereof) or salts of nitrite and/or nitrate. In one particularembodiment beetroot or a juice thereof is administered in order toreduce the blood pressure in a mammal.

The ratio nitrite/nitrate comprising composition:polyphenol-richcompound should be chosen to obtain enough supply of nitrate. Thenitrite/nitrate comprising composition should therefore be at leastabout 10%, preferably at least about 20%, more preferably at least about30%, even more preferably at least about 40% and most preferably atleast about 50% or even more. Examples of polyphenol rich fruit orjuices thereof include, but are not limited to, apple, pear, grapes,lemon, orange, lime, peach, pomegranate, grapefruit, kiwi, ginger andpineapple. Juice from berries are also usable including blackberries,black raspberries, blueberries, cranberries, red raspberries, cherries,bog wortleberry, lingonberries, black elderberry, black chokeberry,black currant, cloudberries and strawberries. Other natural sources ofpolyphenols include vegetables such as carrots, chili, rhubarb, andonions. In addition, cacao products (rich in flavanols), green or blacktea, nuts, Yerba mate and coffee are rich in polyphenols.

In one preferred embodiment, the nitrate in the inventive compositionoriginates from beetroot (such as beetroot juice), which is blended withone or several polyphenol-rich products. The ratio beetrootjuice:polyphenol-rich compound should be chosen to obtain enough supplyof nitrate and therefore the beetroot juice part should be at leastabout 10%, preferably at least about 20%, more preferably at least about30%, even more preferably at least about 40% and most preferably atleast about 50%.

According to one embodiment of the present invention the dose of nitriteand/or nitrate or nitrite and/or nitrate together with polyphenols isadministered or manufactured as a chewing gum, lozenge or pastille,wafer, cake, bar or the like which optionally also comprises livenon-pathogenic bacteria or some other microorganism. Thesemicroorganisms may also be included in “dry form” for example intablets, capsules, bars, and alike. It can also be administered ormanufactured as a functional food product or as a part of a functionalfood product.

Licorice is well known for its blood pressure elevating effects and itis contemplated that the addition of nitrate/nitrite alone or incombination with a polyphenol will counteract this via the NO-mediatedblood pressure lowering effect of these compounds. In particular a saltsuch as potassium nitrate, sodium nitrate or ammonium nitrate may beused to replace in part or in whole the salt content (such as sodiumchloride or ammonium chloride) of the licorice product.

The purpose with the combination with non-pathogenic live bacteria (e.g.the “probiotic” nitrate reducing bacteria) is to further enhance thegeneration of bioactive compounds such as NO, nitroso adducts orchemically related compounds. This enhancement will occur locally in thegastrointestinal (GI) tract via bacteria-dependent reduction of nitrateand nitrite to NO and other bioactive nitrogen oxides. In particular,this combination will be effective in treating and preventing GIdisorders such as ulcers in the stomach, duodenum, jejunum, caecum andcolon/rectum. Also, it is contemplated that this combined product willbe effective in treating and preventing inflammatory bowel disordersincluding ulcerative colitis, Crohn's disease, microscopic colitis andother forms. Irritable bowel disease (IBS) is another condition thatcould be treated with said product. In addition, the compounds formedcan be absorbed systemically and have sustained biological effects forexample in reducing blood pressure and in preventing atherosclerosis,cancer or any other effect related to enhance NO release as discussedabove. The composition with added bacteria can be in the form of a drinksuch as a juice, yoghurt, a milk-based drink or any other fermented foodproduct. The composition with added bacteria can also be included indifferent types of functional food. Suitable bacteria are the so calledprobiotic bacteria, included but not limited to Lactobacilli (forexample, L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius,L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri,L. fermentum, L. reuteri) and Bifidobacteria species (for example, butnot limited to, B. breve, B. bifidum, and B. lactis). Other suitablenon-pathogenic bacteria that enhance nitrate reduction or nitritereduction include e.g. Veillonella species, Staphylococcus species,Actinomyces species, or Rothia species. The microorganisms to be addedto the composition may also be probiotic yeasts (such a Saccharomycesboulardii).

In one embodiment, a low concentration of ethanol is added to theinorganic nitrite and/or nitrate composition. In one embodiment ethanolis used in combination with or administered together with the inorganicnitrite and/or nitrate composition. It has surprisingly been found thatethanol even in very low concentrations can generate the potentvasodilator ethyl nitrite following reaction with physiological amountsof nitrite. The reaction is enhanced at acidic conditions such as in thegastric lumen. It is contemplated that ingestion of nitrate will lead toaccumulation of nitrite in the saliva and the nitrite will react withethanol in the stomach thereby forming ethyl nitrite. For example, ifthe inventive composition is in the form of a liquid the ethanol contentshould be below about 5% (v/v), more preferably below about 2% (v/v),and most preferable between about 0.5-1.5% (v/v).

In one embodiment of the present invention a cacao product such as darkchocolate that is rich in flavanols is combined with a nitrate-richnatural compound in a drink or a chocolate bar. One preferrednitrate-rich compound in this embodiment is rhubarb. Again, the nitratewill potentiate the effect of the flavanols via enhancement of NOformation as described above and in FIG. 1.

Contamination of a nitrate-containing food or drink with unwantedbacteria may result in a large accumulation of nitrite, due to nitratereducing bacterial enzymes. Ingestion of high levels of nitrite maycause potentially serious methemoglobinemia. In one embodiment, anitrate-rich composition is mixed with a compound that inhibits unwantedbacterial growth. Such compound should be chosen so as not to affect thetaste of the product negatively. Ideally, it should enhance the tasteand at the same time increase the bioactivity of the product. One optionis to acidify the inventive composition so that final pH is below about5, and most preferably between about pH 2-4. This will inhibit and/orabolish bacterial growth. Suitable acidifying agents can be any agentthat reduces pH and include artificial compounds as well as naturaljuices from e.g., but not limited to, lemon or lime, ascorbic acid,acetic acid or vinegar (from apple, grapes or other fruits). It iscontemplated that with the use of natural products a dual effect isachieved. Besides having an antibacterial effect, they are rich inpolyphenols, which enhance the generation of bioactive NO fromnitrate/nitrite in the vegetable drink. In one particular embodiment, anitrate-rich vegetable juice (e.g. beetroot juice) is mixed with acompound that inhibits undesirable bacterial growth.

According to an embodiment of the invention, the nitrate and nitritesalts are combined with other pharmaceuticals including but not limitedto: anti-diabetic drugs (insulin and oral anti-diabetics),cardiovascular drugs (statins, ACE inhibitors, beta-receptorantagonists, diuretics, angiotensin 2 receptor antagonists, organicnitrates, calcium channel blockers) acid secretion-inhibitors (protonpump inhibitors, Histamine-2 receptor blockers), oral anti-diabeticsincluding biguanides, sulphonureides, alpha-glucosidase inhibitors,thiazolidinediones, glinides; drugs for treatment of pulmonaryhypertension including prostacyclin analogues, endothelin receptorantagonists and sildenafil.

According to one embodiment the nitrite is delivered systemically viaperoral treatment with an organic nitrate including nitroglycerine orisosorbide mono/di-nitrate. Nitroglycerine is used clinically to treatangina pectoris and it acts by releasing vasodilatory nitric oxidesystemically. However, this drug must be given parenterally because thefirst passage metabolism in the liver is considerable. Interestingly, ithas been found that the liver metabolism of nitroglycerine yieldspredominantly nitrite. Considering the novel biological effects ofnitrite described here, nitroglycerine may therefore be used as a“prodrug” of nitrite. Preferably the drug should then be given by theenteral route (with liver metabolism) to maximize nitrite generationwhile at the same time avoiding the acute vasodilatation and drop insystemic blood pressure associated with iv or sublingual administrationof nitroglycerine. A suitable dose range when giving nitroglycerineperorally is about 0.001 to 10 mol/kg/24 hours, preferably about 0.001to 1 mol/kg/24 hours, more preferably 0.01 to about 0.1 mmol/kg/24hours. The tablets should preferably be coated to avoid absorption inthe oral cavity.

According to one embodiment nitrite and/or nitrate is added toparenteral and enteral feeding/nutrition solutions to be used in adults,children, neonates and prematures. Today such solutions are generallyextremely low in nitrate and nitrite as noted in measurements performedby the present inventors (not shown). An intubated mechanicallyventilated patient with parenteral nutrition is particularly deprived ofnitrate/nitrite. First, these patients do not produce and swallow salivaproperly and thus one great nitrate/nitrite source is disrupted.Secondly as stated above, the feeding they receive contains almost nonitrate/nitrite. Many intensive care patients suffer from metabolicdisturbances, in particular catabolism due to stress and trauma andtheir metabolic rate is often increased. Moreover, insulin resistance iscommon and glucose homeostasis is disturbed. Tight control of plasmaglucose by administering insulin is advocated in these patients. Also,healthy subjects, such as infants or subjects taking enteral solutionsfor other reasons can benefit from the compositions and methods of thepresent invention. Enteral nutrition is thus meant to include alsoinfant formulas and other enteral products.

When given preoperatively, the amount of carbohydrates in the drink willprovide the patient with preferably about 200 kcal, more preferablyabout 100 kcal and most preferably about 50 kcal. For the use of anitrate salt perorally, a dose of about 0.01-100 mmol/kg body weight/24h is currently preferred or more preferably a dose of about 0.01-10mmol/kg body weight/24 h, even more preferably 0.1-1 mmol/kg bodyweight/24 h.

Using intravenous administration pre- and/or peroperatively, the nitriteis preferably administered in a dose within the interval of about 0.01to about 10 000 nmoles/kg body weight/min, preferably about 0.01 toabout 1000 nmoles/kg body weight/min, more preferably about 0.1 to about100 nmoles/kg body weight/min, most preferably about 1 to about 20nmoles/kg body weight/min. It is presently contemplated that the mostpreferred dose is less than about 15 nmoles/kg body weight/min.

Therapeutic Methods of Use

In one embodiment, provided herein are uses of nitrites and/or nitrates,for the manufacture of pharmaceutical products, enteral or parenteralnutritional solutions, preoperative compositions, nutritionalsupplements, or functional food products for administration to bothhealthy persons, such as athletes, or to patients, suffering from one ormore of the conditions exemplified herein. Included in this embodimentare also possible combinations of nitrites and/or nitrates with othercompounds as mentioned above.

In one embodiment, provided herein are methods for the treatment,alleviation and/or prevention of clinical conditions, comprisingadministering an effective amount of a nitrate and/or a nitrite to apatient in need thereof sufficient to treat, alleviate and/or preventsuch condition.

There are many possible clinical conditions where such treatment,alleviation and/or prevention is performed using nitrate and/or nitriteaccording to the present invention:

-   -   glucose control in diabetes/prediabetes    -   metabolic syndrome

The method and composition according to the invention also has generaltherapeutic, alleviating and/or prophylactic effects in patients undermetabolic stress. Examples include but are not limited to:

-   -   intensive care patients    -   patients undergoing surgery    -   patients suffering from malnutrition of different genesis    -   cancer with anorexia    -   burn injury    -   trauma    -   neonates/prematures    -   anorexia nervosa    -   thyroid disorders (e.g. hyperthyreosis/hyperthyroidism)    -   myocardial infarction, cardiac arrest    -   major systemic disease with a catabolic state    -   stress ulcers (gastric)    -   surgical gut anastomosis insufficiency    -   fever [0081] myocardial infarction and cardiac arrest    -   ischemia-reperfusion injury (MI, stroke, arterial insufficiency        or any other organ ischemia)    -   sleep apnea syndrome    -   septic chock    -   insufficient perfusion of the intestines

Patients treated in the ICU (intensive care unit) are often subjected tosevere metabolic stress due to trauma, infection, pain and otherpathological processes. Such patients are treated for several reasonsincluding but not limited to post-surgical observation, trauma,bleeding, burn injury, brain injury, stroke, diabetes, sepsis, septicshock, myocardial infarction, cardiac arrest, arterial insufficiency orany other organ ischemia, chronic obstructive pulmonary disease, asthmaand other severe inflammatory conditions, pulmonary hypertension,congestive heart failure, pulmonary embolism. This results in varyingdegrees of catabolism and resistance to insulin with a disturbed glucosehandling. They also often suffer from vascular endothelial dysfunctionleading to microcirculatory disturbances. As mentioned above thesepatients are given only minute amounts of nitrate/nitrite in the enteraland parenteral feeding and due to several reasons (sedation, intubation)they have a disturbed enterosalivary circulation of nitrate/nitritecompared to healthy people.

It is contemplated that addition of sufficient amounts of nitrite and/ornitrate in the parenteral and/or enteral feeding to patients treated inthe intensive care unit, may alleviate or prevent the aforementionedmetabolic and circulatory disturbances by decreasing the rate ofmetabolism, enhancing blood glucose homeostasis and microcirculatoryimprovement.

Patients undergoing surgery are subjected to a varying degree ofsurgical trauma. This triggers catabolic hormones like cortisol,glucagon and adrenalin and such patients may develop transient insulinresistance. Common clinical procedure involves fasting in order toreduce gastric content, which could be accidentally aspirated in theairways if the patient vomits or regurgitates during anesthesia. Suchfasting eliminates the intake of nitrate/nitrite and it has been shownthat systemic levels of nitrate and nitrite are reduced after fasting.It is contemplated that administration of nitrite and/or nitrate pre,per or postoperatively will improve the metabolic situation (decreasingthe rate of metabolism, enhancing blood glucose homeostasis) in surgicalpatients. Moreover, during the entire perioperative period the patientsare at higher risk for ischemic events due to hypotension, hypoxia andmicrocirculatory disturbances. Such events will initiateischemia-reperfusion processes, which may injure several organ systemswithin the body. It is contemplated that prophylactic administration ofnitrite and/or nitrate, either as a preoperative drink containingnitrate and/or nitrite anions, a suitable vegetable juice such as butnot limited to beetroot juice or by giving nitrate and/or nitrite viathe parenteral route, will reduce the negative effects ofischemia-reperfusion events during surgery in several organs includingbut not limited to brain, heart, lung, liver, kidney and skeletalmuscle.

Ischemia-reperfusion injury during myocardial infarction is a majorproblem in clinical care. State-of-the art treatment of myocardialinfarction includes reopening of occluded coronary vessels bypharmacological means with thrombolytic agents and/or percutaneouscoronary intervention. It is considered that the method and compositionaccording to the invention will reduce ischemia-reperfusion injury byincreasing blood flow and by altering mitochondrial function. Fromexperiments in the present application it is also considered that duringischemia-reperfusion nitrate and nitrite could reduce oxygen consumptionpossibly by a “hibernating” effect on mitochondria. Another mechanism bywhich nitrate and nitrite could ameliorate ischemia-reperfusion injuryis by reducing oxygen radical formation and cytochrome C release fromthe mitochondria. The effect of nitrate and nitrite on mitochondrialfunction is most likely mediated by nitric oxide (NO) interacting withoxidative phosphorylation and/or s-nitrosylation of protein complexes inthe mitochondrial respiratory chain. It is envisaged that the time pointfor administration of nitrate and/or nitrite in relation to themyocardial infarction induced ischemia-reperfusion injury will beeffective both pre, per or post injury. Both a pre- andpost-conditioning effect of nitrate and/or nitrite is considered whichmeans that a patient can receive treatment already in the ambulance onhis/her way to the hospital, at the hospital before and afterreperfusion of the coronary circulation and also at the ward. Also,patients at high risk of developing a myocardial infarction (e.g. anginapectoris, congestive heart failure) could be considered for prophylactictreatment with the method and composition according to the invention.After cardiac arrest and resuscitation systemic ischemia-reperfusioninjury develops involving several major organ systems, including thebrain. It is contemplated that nitrate and/or nitrite is beneficial bothgiven prophylactic to patients with high risk for cardiac arrest andalso during resuscitation procedures.

According to a preferred embodiment, the nitrate and/or nitrite or anycombination mentioned herein is/are also given pre-operatively topatients scheduled to undergo surgery or substantive, invasiveexamination procedures. In one particular embodiment, inorganic nitriteand/or nitrate are combined with carbohydrates. Thus, such combinationsinclude, but are not limited to nitrate alone; nitrate and nitrite;nitrate, nitrite, and carbohydrates; nitrate and carbohydrates; nitritealone and nitrite and carbohydrates. In addition, polyphenols may beadded to any of the aforementioned combinations. Any such combinationcan be administered (in single or repeated doses) as a preoperativedrink or the like prior to surgery or intravenously prior or duringsurgery. An appropriate time to administer such a preoperative drink isbetween 72 and 2 hours before surgery. A combination of nitrite and/ornitrate with carbohydrates may ameliorate insulin resistance and alsoischemia/reperfusion injury common in the preoperative and postoperativeperiod. Examples of carbohydrates include but are not limited toglucose, fructose, maltodextrin, sucrose, lactose, galactose andmannose.

In patients suffering from arterial insufficiency, including but notlimited to intermittent claudication, pharmacological treatment aims atimproving blood flow in the affected limb(s) and to stimulateangiogenesis to promote new vessel formation.

Phosphodiesterase inhibitors and growth factors have been studied inclinical trials but results are variable. Likewise, studies withtraditional NO donors such as organic nitrates have been lesssuccessful. It is contemplated that the method and composition accordingto the invention will positively stimulate both blood flow andangiogenesis thereby improving the condition in these patients.Treatment with nitrate/nitrite is attractive since it willpreferentially increase blood flow in the ischemic areas without causingtroublesome systemic effects such as hypotension, which is a risk whenusing organic nitrates that dilate non-selectively in most vascularbeds. In addition, nitrite will affect mitochondrial function asdiscussed above resulting in less oxygen demand in the ischemic tissue.

In patients suffering from malnutrition, including but not limited tocancer with anorexia, anorexia nervosa, gastrointestinal disease, it iscontemplated that the method and composition according to the inventionwill have an anabolic effect and will reduce oxygen consumption. It iscontemplated that this effect is achieved by improving mitochondrialefficiency and attenuating mitochondrial uncoupling.

The method and composition according to the invention also has generaltherapeutic effects in patients with erectile dysfunction. In thiscondition, the endogenous NO system is failing. Administration ofnitrite and/or nitrate (and possible combinations outlined above) willenhance NO formation locally in the corpus cavernosum, thereby enhancingerection.

It is contemplated that the method and composition according to theinvention has general therapeutic, alleviating and/or prophylacticeffects in patients with gastritis and gastric ulcers due toHelicobacter pylori infection, stress or side effects frompharmacological treatment such as the use of Non-SteroidalAnti-Inflammatory Drugs (NSAID). By improving gastric mucosal bloodflow, mucus generation and by anti-bacterial and anti-inflammatoryproperties the method and composition will have beneficial effects inthese patients. In addition, unwanted side effects of treatment withacetylsalicylic acid, NSAIDs or any other ulcerogenic drug in otherparts of the gastrointestinal tract (duodenum, small and largeintestines) are prevented by said method and composition.

A feared problem in surgery is gut anastomosis insufficiency andinsufficient perfusion of the intestines. New methods for improvingmicrocirculation in the anastomosis are constantly investigated. It iscontemplated that the method and composition according to the inventionwill improve microcirculation in the anastomosis, which will enhance thehealing process. It is also considered that the invention will improvecirculation in situations with insufficient perfusion of the intestines.

Patients with sleep apnea syndrome are at higher risk for developinghypertension and other cardiovascular diseases. Moreover, they aresubjected to periods of hypoxia during sleep. It is contemplated thatthe method and composition according to the invention will protectagainst hypoxia-induced injury by improving circulation and by reducingoxygen consumption possibly by a “hibernating” effect on mitochondria.Other mechanism by which said method and composition could haveprotective effects is by reducing oxygen radical formation andcytochrome C release from the mitochondria. The effect of nitrate andnitrite on mitochondrial function is most likely mediated by nitricoxide (NO) interacting with oxidative phosphorylation and/ors-nitrosylation of complexes in the mitochondrial respiratory chain.

The method and composition according to the invention has generaltherapeutic, alleviating and/or prophylactic effects also in patientswith pathological conditions characterized by low oxygen availability,including but not limited to chronic obstructive airway disease (COPD),inflammatory airway disease such as asthma, pulmonary hypertension,congestive heart disease, interstitial lung disease, pulmonary embolism,ischemic heart disease, peripheral artery disease, sleep apnea syndrome,myocardial infarction and systemic inflammatory disorders. In theseconditions, addition of oxygen promptly improves arterial oxygenationand total body oxygen delivery. For technical and safety reasons, it iscomplicated to administer oxygen outside hospital facilities. Anotherway to improve the situation for these patients is to reduce the needfor oxygen. The method and composition according to the invention leadto reduced oxygen cost in relation to the physical work performed. Thishighly surprising finding is especially relevant in the patients withthe aforementioned conditions since oxygen availability is the limitingfactor for physical activity. It is envisaged that the method andcomposition according to the invention will facilitate physical activityin these patient groups.

In one preferred embodiment nitrate and/or nitrite is given to patientswith a pathological condition characterized by low oxygen availability.In such situations, it is desirable to reduce the tissues need foroxygen to prevent sequele and symptoms associated with the hypoxia.Examples include patients with COPD whose pulmonary oxygen uptake may beseverely compromised and patients with peripheral artery disease whereoxygen delivery to the tissues is reduced.

Non-Therapeutic Effects and Methods of Use

The inventive method and composition is also useful for healthysubjects, e.g. athletes. The inventive method and composition providesfor less oxygen demand at a certain workload and improves anabolism. Themethod and composition is also useful for oxygen sparing at highaltitude, for example in work and sports performed in a low oxygenenvironment, such as but not limited to rescue activities, firefighting,military operations, diving, mountain climbing, high-altitude flying,and the exploration of space.

The present method and composition is also useful in solid organ ortissue transplantation, in order to minimize metabolic demand of thedonated organ before transplantation, and to improve survival of thetransplanted organ or tissue after transplantation. Nitrite and/ornitrate or any combination mentioned herein is/are given either into theorgan or tissue by perfusion, topically on the organ or tissue and/orsystemically to the donor before transplantation, and into the organ ortissue by perfusion or topically on the organ or tissue and/orsystemically to the receiver after transplantation.

In one preferred embodiment nitrate and/or nitrite is/are or anycombination mentioned herein given to patients that are at risk ofdeveloping significant metabolic stress. Such situations include many ofthe conditions listed above including surgical stress and trauma. Oxygendemand and consumption increases dramatically during stress. Thus, animproved situation is achieved by decreasing the oxygen demand in thesepatients. According to one preferred embodiment nitrate and/or nitriteis given to patients to prevent the sequele associated with physiologicor traumatic stress. Examples include patients that come into theemergency room after trauma or patients undergoing major surgery.

To the best of the knowledge of the inventors, this is the first studyto examine the effects of dietary nitrate on the cardiopulmonary andmetabolic response to exercise. The main finding was that dietarysupplementation with inorganic nitrate, in an amount which does notcause significant hypotension and without any significant increase inmethemoglobin and plasma lactate, results in a reduced VO₂ duringsubmaximal work and a significant increase in muscular efficiency. Theseeffects occurred without any changes in VO₂peak values or maximalattainable work rate. Thus, the present invention also makes available asecond non-medical use of inorganic nitrate and/or nitrite, i.e. for themanufacture of a composition for enhancing the performance of a mammalwherein the effect of enhancing performance is manifested as reduced VO₂during physical exercise. Preferably the effect of enhancing performanceis manifested as both a reduced VO₂ during physical exercise and asignificant increase in muscular efficiency.

Accordingly, in one implementation, the inventors make available amethod for non-therapeutically enhancing the performance of a mammal,wherein inorganic nitrate and/or nitrite is administered to said mammal.Said mammal is chosen among a human, a horse, or a dog, preferably ahuman.

According to one particular embodiment, only nitrite is administered.

Without wishing to be bound by theory, the inventors consider that thereis reason to believe that the observed effects involve initial reductionof nitrate to nitrite. Nitrate itself is believed to be biologicallyinert and cannot be metabolized by mammalian cells. However, afteringestion nitrate re-enters into the mouth via the salivary glands andis effectively reduced by commensal bacteria thereby forming nitrite. Incontrast to nitrate the nitrite ion has recently been shown to possess awide range of bioactivities. In the present study, the inventors didindeed note an increase in plasma nitrite after the nitrate treatmentperiod thereby confirming in vivo reduction of nitrate as describedpreviously. Another finding in support of nitrite being bioactive wasits effective consumption during exercise in contrast to the unchangedlevels of plasma nitrate. Ultimately the bioactivity of nitrite islikely related to its further reduction to NO and possibly other closelyrelated nitrogen intermediates. In addition, it has been recentlysuggested that, nitrite itself may directly affect cellular signalingpathways. Although probably unlikely, at this stage effects of thenitrate ion itself cannot be excluded. There are several principle waysby which biological effects of nitrogen oxides may be propagatedincluding activation of cGMP, alteration of protein function by anitro(syl)ation/nitration or direct binding to protein heme-moieties ofseveral proteins as in the prototypic activation of guanylyl cyclase byNO. In addition, nitrite itself may also directly affect cellularsignaling pathways.

If the effects proceed via nitrate reduction to nitrite and then NOformation, how could this then explain the present results? Earlierstudies using NOS inhibitors to block endogenous NO production give someindications. NOS-inhibition has been shown to increase submaximal VO₂ indogs during exercise, independently of the reduction in blood flow. Thisincrease in VO₂ during NOS-blockade has been linked to the fact that NOaffects tissue respiration in vitro by reversible inhibition of therespiratory enzyme cytochrome c oxidase. Others have related theincreased VO₂ during NOS-blockade to an inhibiting effect of NO onproton leakage via the mitochondrial permeability transition pore (mPTP)were a considerable amount of protons leak over the inner mitochondrialmembrane. If the effects of nitrate were solely due to inhibition ofcytochrome c oxidase (thereby inhibiting oxidative phosphorylation) onewould expect an increase in anaerobic metabolism during physicalexercise and a larger accumulation of lactate. However, judging from theresults this was not the case, as the plasma lactate concentration wasnear identical after nitrate supplementation compared to placebo.

The finding that the oxygen pulse at a given work rate decreases bynitrate supplementation is a direct effect of the lower oxygen demand atthat work rate. However, there is no difference in oxygen pulse at agiven absolute oxygen uptake. The lack of effect of nitrate on VE/VO₂ oroxygen pulse indicates that the improved efficiency originates frommuscular or mitochondrial adaptations rather than from centraladaptations in the heart or the lungs.

There is reason to believe that the observed effects of nitrate onphysical performance involve initial reduction of nitrate to nitrite.Nitrate itself is believed to be biologically inert and cannot bemetabolised by mammalian cells. However, after ingestion nitratere-enters into the mouth via the salivary glands and is effectivelyreduced by commensal bacteria thereby forming nitrite. In contrast tonitrate the nitrite ion has recently been shown to possess a wide rangeof bioactivities.

The inventors noted an increase in plasma nitrite after the nitratetreatment period thereby confirming in vivo reduction of nitrate asdescribed previously (Lundberg & Govoni 2004, LARSEN, F J, et al.Effects of dietary nitrate on blood pressure in healthy volunteers. NEngl J Med. 2006, vol. 255, no. 26, p. 2792-3). Another finding insupport of nitrite being bioactive was its effective consumption duringexercise in contrast to the unchanged levels of plasma nitrate.Ultimately the bioactivity of nitrite is likely related to its furtherreduction to NO and possibly other closely related nitrogenintermediates. In addition, it has been recently suggested that nitriteitself may directly affect cellular signalling pathways (BRYAN, N S, etal. Nitrite is a signaling molecule and regulator of gene expression inmammalian tissues. Nat Chem Biol. 2006, vol. 1, no. 5, p. 290-7).Although probably unlikely, at this early stage, effects of the nitrateion itself cannot be excluded. There are several principle ways by whichbiological effects of nitrogen oxides may be propagated includingalteration of protein function by nitrosylation, nitration or directbinding to protein heme-moieties as in the prototypic activation ofguanylyl cyclase by NO.

Earlier studies using NOS inhibitors to block endogenous NO productiongive some indications. NOS-inhibition has been shown to increasesubmaximal VO₂ in dogs during exercise, independently of the reductionin blood flow (SHEN, W. et al. Nitric oxide. An important signalingmechanism between vascular endothelium and parenchymal cells in theregulation of oxygen consumption. Circulation. 15 Dec. 1995, vol. 92,no. 12, p. 3505-12., ISHIBASHI, Y, et al. ATP-sensitive K+-channels,adenosine and NO-mediated mechanisms account for coronary vasodilationduring exercise. Circulation Res. 1998, no. 82, p. 346-359.; Shen et al.1999, supra). The increase in VO₂ during NOS-blockade has been explainedby the fact that NO affects tissue respiration by reversible inhibitionof the respiratory enzyme cytochrome c oxidase (Carr & Ferguson 1990,supra; Bolanos et al. 1994, supra; Brown & Cooper 1994, Cleeter et al.1994, Schweizer & Richter 1994). Others have related the increased VO₂during NOS-blockade to an inhibiting effect of NO on proton leakage overthe inner mitochondrial membrane (BOHUSLAVS′KYI, A, et al. Effect ofnitric oxide on the efficiency of oxygen consumption by the workingskeletal muscle in fatigue. Fiziol Zh. 2005, vol. 51, no. 1, p. 33-42;NAVET, R, et al. Proton leak induced by reactive oxygen species producedduring in vitro anoxia/reoxygenation in rat skeletal musclemitochondria. J Bioenerg Biomembr. 2006, vol. 38, no. 1, p. 23-32; WANG,G, et al. Nitric oxide donors protect murine myocardium againstinfarction via modulation of mitochondrial permeability transition. Am JPhysiol Heart Circ Physiol. 2005, vol. 288, no. 3, p. 1290-5). If theeffects of nitrate were solely due to inhibition of cytochrome c oxidaseone would expect an increase in anaerobic metabolism during physicalexercise and a larger accumulation of lactate. However, judging from theresults this was not the case, as the plasma lactate concentration wasnear identical after nitrate supplementation compared to placebo. Theinventors consider this to be very surprising.

The studies using NOS inhibitors cited above all imply that endogenousNO is involved in regulation of oxygen consumption but there have beenfew attempts to study the effect of exogenous NO delivery. Studies withNO-donors such as nitroprusside and nitroglycerine have shown somewhatdiverging results, with decreases in VO₂ in some cases (RECCHIA, F A, etal. Nitric oxide controls cardiac substrate utilization in the consciousdog. Cardiovasc Res. 1999, no. 44, p. 325-32; LOKE, K E, et al. Nitricoxide modulates mitochondrial respiration in failing human heart.Circulation. 21 Sep. 1999, vol. 100, no. 12, p. 1291-7), no effect inone study (NUNEZ, C, et al. Discrepancies between nitroglycerin andNO-releasing drugs on mitochondrial oxygen consumption, vasoactivity,and the release of NO. Circ Res. 11 Nov. 2005, vol. 97, no. 10, p.1063-9) and increases in other settings (DE BACKER, D, et al. Effects ofdobutamine on the relationship between oxygen consumption and deliveryin healthy volunteers: comparison with sodium nitroprusside. Clin Sci(Lond). 1996, vol. 90, no. 2, p. 105-11).

Several of the proposed mechanisms for nitrite reduction to NO describedabove could theoretically come into play during physical exercise. Thus,all these pathways are greatly enhanced during hypoxia and when pHdecreases such as in a working muscle. Shiva and colleagues veryrecently demonstrated deoxymyoglobin-dependent nitrite reduction to NOin rat heart homogenates with a concomitant inhibition of mitochondrialrespiration (Shiva et al 2007, supra). Another possible pathway includesNO formation by the mitochondria themselves (KOZLOV, A V, et al. Nitritereductase activity is a novel function of mammalian mitochondria. FEBSLett. 2 Jul. 1999, vol. 454, no. 1-2, p. 127-30) or even simple acidicreduction of nitrite in the working muscle (Zweier et al. 1995, supra,MODIN, A, et al. Nitrite-derived nitric oxide: a possible mediator of‘acidic-metabolic’ vasodilation. Acta Physiol Scand. 2001, vol. 171, no.1, p. 9-16). Cosby and colleagues described NO formation andvasodilation from the reaction of circulating nitrite ions withdeoxyhemoglobin in blood (COSBY, K, et al. Nitrite reduction to nitricoxide by deoxyhemoglobin vasodilates the human circulation. Nat. Med.2003, vol. 9, no. 12, p. 1498-505). While this latter pathway, andpossibly also tissue nitrite reduction, very well might explain therecently described nitrate-induced reduction in resting blood pressure(Larsen et al. 2006), it is still not obvious how this NO also woulddecrease oxygen consumption in the working muscle. Thus, an effectiveinhibition of mitochondrial respiration e.g. by deoxymyoglobin-derivedNO, would again be expected to result in accumulation of plasma lactatewhich was not the case.

The efficiency of the muscles to produce work has been related to thepercentage of type I muscle fibres (COYLE, E F, et al. Cyclingefficiency is related to the percentage of type I muscle fibers. Med SciSports Exerc. 1992, vol. 24, no. 7, p. 782-8) and uncoupling protein-3(UCP3) content of muscle fibres (MOGENSEN, M, et al. Cycling efficiencyin humans is related to low UCP3 content and to type I fibers but not tomitochondrial efficiency. J Physiol. 2006, vol. 571, no. 3, p. 669-681).Other factors that might contribute to the efficiency of movement areanatomical, biochemical and biomechanical features (WILLIAMS, K R. Therelationship between mechanical and physiological energy estimates. MedSci Sports Exerc. 1985, no. 17, p. 317-25). Thus, simply measuringdifferences in VO₂ at different work rates is not an optimal estimate ofmuscular efficiency because the energy output for a certain VO₂ isdependent upon substrate utilization. Gross efficiency (GE) calculationsinclude possible changes in respiratory exchange ratio and thereby takesubstrate utilization into account. The improved GE after nitratesupplementation indicates better efficiency, but even so, it cannot beexcluded that this improved efficiency originates from reduced baselineenergy expenditure (EE). The Delta efficiency (DE) calculations are notdependent on the baseline EE and are also based on all work rates takentogether instead of a single work rate at a time as in theGE-calculations. It is therefore plausible to expect DE to be the mostvalid estimate of muscular efficiency in this case. Indeed, even DE wassignificantly improved after nitrate supplementation. It is unlikelythat the improved efficiency by nitrate comes from mechanical factors.The subjects of this study were all cyclists with many years ofexperience of training and competing. It is improbable that a few visitsto the laboratory would change their efficiency during cycling to anynoteworthy extent. Especially since the subjects used the same cyclingshoes, clip-on pedals and the same seat position as they were used toduring training makes this even more unlikely. More important, therandomization procedure used in this study rules out any suchdifferences. Marcheal and Gailly (MARCHEAL, G, et al. Effects of nitricoxide on the contraction of skeletal muscle. Cell Mol Life Sci. 1999,no. 55, p. 1088-1102) demonstrated a faster relaxing velocity of musclefibres in in situ experiments during administration of an NO-donor,thereby implicating a neuromuscular modulatory effect of NO. It remainsto be proven if this can improve the muscular efficiency during cycling.

The finding that the oxygen pulse at a given work rate decreases bynitrate supplementation is a direct effect of the lower oxygen demand atthat work rate. However, there is no difference in oxygen pulse at agiven absolute oxygen uptake. The lack of effect of nitrate on VE/VO₂ oroxygen pulse indicates that the improved efficiency originates frommuscular or mitochondrial adaptations rather than from centraladaptations in the heart or the lungs.

In summary, the present findings demonstrate a lower oxygen cost duringsubmaximal work after dietary supplementation with nitrate, in amountsachievable through the intake of a non-toxic amount of nitrite. Thisoccurred without an accompanying increase in plasma lactate, indicatingthat the energy production had become more efficient. The mechanism ofaction and main targets need to be clarified but the process likelyinvolves in vivo reduction of nitrate into bioactive nitrogen oxidesincluding nitrite and NO.

EXAMPLES

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionas set forth in the claims appended hereto.

The present inventors have been studying the effects of nitrate andnitrite on various physiological functions including blood pressure,glucose metabolism and energy expenditure in vivo. In the examplesbelow, unless otherwise noted, nitrate was administered perorally andnitrite was administered intravenously (iv). Oxygen consumption wasmeasured using indirect calorimetry.

1. Intravenous Sodium Nitrite and Resting Energy Expenditure

Resting energy expenditure (as measured by indirect calorimetry) isreduced by 10-25% after 10 min of iv infusion of sodium nitrite (n=4,FIG. 3). Prior to the test the subjects had been on a diet low innitrate for one day and had been fasting for at least 8 h. The majordrop in energy expenditure was seen when infusing nitrite at aconcentration of 10 nmoles/kg body weight/min during 10 minutes. At 1nmol/kg body weight/min no obvious effects were noted during the 10 minobservation period and after 100 nmol/kg body weight/min no furtherdecrease in oxygen consumption was noted as compared to the 10 nmoldose. In separate experiments (n=2) the present inventors noted anincrease in plasma nitrite from 140-165 nM to 370-480 nM after the 10nmol/kg body weight/min infusion (10 min infusion). Basal levels ofmethemoglobin were 1.1-1.3 mmol/1 and did not change significantly afterinfusion of nitrite (1.1-1.4 mmol/1) See FIG. 2.

2. Oral Sodium Nitrate and Oxygen Consumption During Exercise

A. Methods

Subjects: Nine healthy, well trained (VO₂peak 55±3.7 ml×kg⁻¹×min⁻¹),males (28±6 years) volunteered for the study. All subjects were trainedcyclists or triathletes and well accustomed to the testing procedure. Itwas chosen to use well-trained subjects to avoid training effects fromthe tests such as enhanced VO₂peak or better mechanical efficiencyduring submaximal exercise. The protocol was approved by the regionalethics committee in Stockholm and all subjects gave their writtenconsent prior to participation.

Dietary supplementation with nitrate: The aim with the present study wasto investigate the effects of two distinct dietary patterns, one withhigher, and one with lower than normal nitrate intake. The study had adouble-blind placebo-controlled crossover design. During two three-dayperiods, separated by a washout interval of ten days, the subjects wereinstructed to avoid all foods with moderate or high nitrate content (allvegetables, all cured meats, strawberries, grapes, and tea). Inaddition, they were told to restrain from alcohol and tobacco products.Otherwise they were free to eat any food they liked during the threedays of restricted diet. The subjects were randomized to start witheither ingestion of 0.1 mmol sodium nitrate/kg body weight/day dissolvedin water or an equal amount of sodium chloride (placebo). The daily dosewas divided and ingested three times daily. The different solutionscould not be distinguished by taste or appearance. The daily nitratedose corresponded to the amount normally found in 150-250 gram of anitrate-rich vegetable such as spinach, lettuce or beetroot. The lastdose of nitrate or placebo was ingested in the morning on the day ofmeasurement (see the main tests below). The order between the nitratesupplementation period (NIT) and the placebo period (CON) was balanced.During the washout period the subjects did not adhere to any specificdietary regime.

Experimental protocol: Measurements were carried out on an electricallybraked cycle ergometer (Monark 839E, Varberg, Sweden) that was modifiedwith a racing saddle and the pedal system the subjects were familiarwith from training. The bicycle ergometer was computer-controlled,permitting a constant work rate regardless of the cadence the subjectchose to pedal with. The pedaling cadence was individually chosen in therange of 70-90 rpm but kept constant during the test to minimizedifferences in work output due to changes in muscle recruitmentpatterns.

Pulmonary ventilation (V_(E)), oxygen uptake (VO₂), CO₂ output (VCO₂)and respiratory exchange ratio (RER) were measured at 10 secondintervals by a computerized gas analyzer (AMIS 2001, Odense, Denmark)connected to a flow meter which the subjects breathed through via amouthpiece and a plastic tube. Heart rate (HR) was continuously recordedduring the tests with a portable heart rate monitor (Polar 5610, Polar,Kempele, Finland). Capillary blood samples (20 μl) were collected fromthe fingertip and were analyzed for lactate ([Hla]) using a BiosenC-Line Sport Analyser (EKF diagnostics, Magdeburg, Germany). Haemoglobinconcentration ([Hb]) at rest was determined with capillary blood takenfrom the fingertip and analyzed with an Hb-measuring device (Hemocue,Angelholm, Sweden). Hematocrit (Hct) was determined by centrifugingcapillary blood at 12000 rpm for three minutes.

Pre-tests: Each subject attended the laboratory twice within a two-weekperiod before the first main tests. The first pre-test was carried outto familiarize the subject with the bicycle ergometer and the testingprocedure. The subjects did a preliminary test at five submaximal levelswith every level lasting for five minutes. There was no rest between thedifferent submaximal levels. VO₂ was continuously measured with the AMIS2001. At the end of each submaximal level capillary blood was taken fromthe fingertip and later analysed for [Hla]. At every work rate thesubjects rated their perceived exertion on the Borg's RPE-scale (BORG,G. Perceived exertion as an indicator of somatic stress. Scand J RehabilMed. 1970, vol. 2, no. 2, p. 92-8), both central and muscular exertionswere rated. After eight minutes of recovery, the subject was instructedto cycle for as long as possible at a work rate corresponding to hiscalculated maximal oxygen uptake (ASTRAND, P-O, et al. Textbook in workphysiology. McGraw-Hill, 1970. ISBN 0070024065. p. 619). During thistest the subjects actual VO₂peak was measured and if the subject wasable to cycle for longer than seven minutes extra power of 20-30 wattswas added every minute until exhaustion. One and three minutes after themaximal test capillary blood were sampled from the fingertip foranalysis of [Hla].

Before the second pre-test, the submaximal levels were adjusted so thatthey corresponded to 45, 60, 70, 80 and 85% of VO₂peak. The maximal workrate was also adjusted, if necessary, so that the time to exhaustion waskept between four and seven minutes.

The main tests: The subjects refrained from heavy exercise three daysprior to the main tests and avoided all exercise the day before thetests. They were also told to eat their last light meal at least 3 hoursbefore the start of the tests. When the subjects came to the laboratorythey received their last dose of either placebo or nitrate and wereallowed to rest in the supine position for 60 minutes before the testcommenced.

All subjects used a standardized warm up procedure of five min ofcycling at 100 watts followed by five minutes of rest. The submaximaland maximal tests were performed in the same way as the second pre-testwith five submaximal work rates lasting five minutes each, without restbetween the different levels. Identical work rates were used during thetwo main tests. Venous blood (9 ml) was drawn at rest 45 minutes afterthe last nitrate/placebo-dose was ingested and again immediately afterthe VO₂peak-test. The blood was placed in an ice bath and centrifugedwithin five minutes at 1300 rpm and 4° C. The plasma was separated andkept at −80° C. until it was analyzed for its nitrate and nitriteconcentrations by a chemiluminescence assay as described previously(LUNDBERG, J O, et al. Inorganic nitrate is a possible source forsystemic generation of nitric oxide. Free Rad Bio Med. 2004, vol. 37,no. 3, p. 395-400).

Statistics and calculations: Results are expressed as means±standarddeviation (mean±SD). Paired t-tests were used to evaluate the differencebetween the nitrate and the placebo trials. The significance level wasset as p≤0.05.

Gross efficiency (GE) was defined as the work rate divided by the actualenergy expenditure (EE). The EE was in turn calculated with the Brouwerequation (BROUWER, E. On simple formulae for calculating the heatexpenditure and the quantities of carbohydrate and fat oxidized inmetabolism of men and animals, from gaseous exchange (Oxygen intake andcarbonic acid output) and urine-N. Acta Physiol Pharmacol Neerl. 1957,no. 6, p. 795-802). Delta efficiency (DE) was defined as the increase inwork rate divided by the increase in EE (GAESSER, G A, et al. Muscularefficiency during steady-rate exercise: effect of speed and work rate. JAppl Physiol. 1975, no. 38, p. 1132-1139). The DE was based on the fourlowest work rates and was analyzed with linear regression. The oxygenpulse is defined as VO₂/HR.

B. Results

Blood pressure at rest: Average resting systolic blood pressure waslower after nitrate supplementation (112±8 mmHg) compared to placebo(120±5.9, p<0.01). The diastolic blood pressure was also lower afternitrate (68±5.5 mmHg) compared to placebo (74±6.8 mmHg, p<0.01). Partsof these findings have been published as a separate communication(Larsen et al. 2006).

Blood values: No change was observed in [Hb] at rest (NIT 152±11, CON153±11 g×l⁻¹, p=0.87) or immediately after the VO₂peak-test (NIT 163±13,CON 161±13 g×l⁻¹, p=0.27). Nor were there any change in the hematocritvalue at rest (NIT 42±4, CON 43±3%, p=0.19) or after the VO₂peak-test(NIT 46±4, CON 47±4%, p=0.6).

Plasma levels of nitrate at rest were 27±6.9 μM in CON and 182±55 in NIT(p≤0.01). Nitrate levels immediately after the maximal work test were29±6.1 in CON and 175±61 μM in NIT (p≤0.01). Plasma nitrate did notchange during exercise either in NIT or in CON (p=0.8). Nitrite levelsat rest were 124±28 in CON and 226±87 nM in NIT (p≤0.01). Immediatelyafter the maximal work test the nitrite levels were 111±29 in CON and137±48 in NIT (p=0.17).

The decrease in nitrite concentrations during exercise was morepronounced in NIT than in CON (FIG. 6). The increase in c-GMPconcentrations after the maximal work as compared to rest tended to behigher in NIT than in CON (p=0.08).

Blood pressure: Average resting systolic blood pressure decreased from120+/−5.9 after NIT to 112±8 mmHg after CON (p=0.003). The diastolicblood pressure decreased from 74±6.8 to 68±5.5 mmHg in the CON andNIT-groups respectively (p=0.005). Parts of these findings have beenpublished as a separate communication (LARSEN, F J, et al. Effects ofdietary nitrate on blood pressure in healthy volunteers. N Engl J Med.2006, vol. 355, no. 26, p. 2792-3).

Submaximal work parameters: After nitrate administration VO₂ wassignificantly lower during the four work rates corresponding to 45-80%VO₂peak compared to the placebo period (FIG. 5). The most significantdifference was seen at 80% of VO₂peak (NIT 3.44±0.31 l×min⁻¹ vs CON3.61±0.31 l×min⁻¹, p=0.003, FIG. 5). On average VO₂ was 0.15 l×min⁻¹lower in the NIT-trials over the 4 submaximal work rates. There was nodifference in heart rate (HR) between the NIT and CON-trials (see FIG.6). The oxygen pulse tended to decrease from 21.0±2.0 during CON to20.3±1.9 ml×beat⁻¹ (p=0.08). No significant differences changes werefound between NIT and CON in [Hla] (FIG. 6), V_(E), V_(E)/VO2 orrespiratory exchange ratio (RER) during any of the submaximal workrates. The average Gross efficiency (GE) is defined as the work ratedivided by the actual energy expenditure (EE). The EE was in turncalculated with the Brouwer equation (Brouwer, supra). GEgrossefficiency improved from 19.7% during CON to 21.1% during NIT (p=0.02).Delta efficiency (DE) is the increase in workload divided by theincrease in EE (Gaesser & Brooks, supra). The DE is in this case basedon the four lowest work rates, which were analyzed with a linearregression analysis. The DE Delta efficiency (DE) increasedsignificantly from 22.1±1.6% during CON compared to 22.9±1.9% duringNIT, (p=0.04).

Maximal work capacity: There was no significant difference in theVO₂peak between the NIT and CON trials (4.49±0.44 and 4.61±0.28 l×min⁻¹respectively, p=0.29). These values were also not significantlydifferent from the VO₂peak achieved during the pre-test (4.54±0.32l×min⁻¹). Likewise, no significant differences were noted either inV_(Emax) (NIT 182±21.4 vs CON 186±21.7 l×min⁻¹, p=0.5), HR.sub.max (NIT189.8±7.0 vs CON 190.3±7.5 beats×min⁻¹, p=0.94) or maximal work rate(NIT 360.6±32.8 vs CON 358.9±32.3 watt, p=0.35). There was no differencebetween NIT and CON in the rating of perceived exertion (Borg RPE-scale)at any work load (submax or max).

Comment to the results: In the present study a significantly reducedoxygen demand at submaximal workloads was noted after nitrateadministration was noted at the four lowest workloads. The fifth workrate, at approximately 85% VO₂peak, was well above the lactate thresholdin several subjects and thus the anaerobic energy production became morepronounced. This led to involvement of accessory muscle groups and anoticeably change in motion pattern. At this work rate the VO₂ did notreach a stable steady-state level and is therefore unsuitable for thecalculation of muscular efficiency. The reason for including this fifthwork rate in the protocol was to receive a lactate value above thelactate threshold and thereby get an indication of changes in the upperpart of the lactate curve.

3. Oral Sodium Nitrate and Glucose Homeostasis

The increase in plasma glucose after a standard Oral Glucose ToleranceTest (75 g glucose in 250 ml water) is lower when measured after nitratepre-treatment (1 mmol/kg body weight NaNO₃) compared to placebo (NaCl).The study was performed with three healthy test subjects in adouble-blind study. Glucose ingestion started 60 min after the nitrateingestion. Blood glucose levels were measured during the 120 min periodafter the glucose load (n=3). At 30 min mean plasma glucose was 8.2mmol/1 with placebo and in the same subjects 6.5 mmol/1 after nitratesupplementation. The results are shown in FIGS. 7a, b , and c.

The lower increase in plasma glucose after nitrate pre-treatment wasverified in a further study wherein a standard Oral Glucos ToleranceTest (75 g glucose) was performed in 8 healthy non-smoking subjectsafter a three-day supplementation with either sodium chloride or sodiumnitrate at equal molar amounts (0.1 mmol/kg body weight/day). The studyhad a double-blind, placebo-controlled, cross-over design. Blood glucoselevels were measured at two time points before (−10 and 0 min) and at15, 30, 45, 60, 75, 90, 105 and 120 min after glucose intake (n=8). Atthe occasion when the subjects had taken nitrate, the area under thecurve for blood glucose was smaller compared to when they had ingestedplacebo. The results are shown in FIG. 8 as mean±SEM.

4. Effect of Ingestion of Beetroot on Blood Pressure

A 43-year-old non-smoking subject with hypertension ingested freshbeetroot juice (300-400 ml/day) for 14 days. The blood pressure wasmeasured twice a day for 14 days and then twice on day 20 (counted fromday 1 of treatment). The Basal blood pressure was 142/99 on the day whenthe experiment started.

The results from the ingestion of beetroot juice are shown in FIG. 9.The mean of the two daily measurements are shown in the bars. Withingestion of the beetroot juice (between day 1-14) a mean reduction insystolic pressure of ≅15 mmHg and a mean reduction in diastolic pressureof ≅16 mmHg was seen. When measured again 6 days after stopping beetrootjuice treatment (day 20), the blood pressure had increased to basallevels (140/100). Pulse rate was unchanged throughout the experimentalperiod. MAP=mean arterial pressure.

5. Concentrations of Plasma Nitrite after Intravenous Infusion ofNitrate

A. Methods

Anesthetized rats (n=11) were given a bolus dose (10 mg/kg body weight)of NaNO.sub.3 (open boxes) and blood samples were collected at indicatedtime points. 7 additional rats were pre-treated with 30 mg/kg bodyweight of allopurinol given intra peritoneally 60 minutes prior to theNaNO.sub.3 infusion (closed triangles) and blood samples were collectedat the time points indicated. Allopurinol inhibits the xanthine oxidase,an enzyme suggested being involved in the reduction of nitrate tonitrite in mammal cells. Plasma was extracted and analyzed for nitrateand nitrite.

Three different strains of mice—wild type (n=5 placebo, n=5 nitrate),germ free (n=5 placebo, n=5 nitrate) and eNOS knockout mice (n=2placebo, n=3 nitrate), were given an intra peritoneal injection of 10mg/kg body weight nitrate or placebo (NaCl) and plasma level of nitritemeasured 1 hour later.

B. Results

The results from the intravenous infusion of nitrate are shown in FIG.10 a-c. FIG. 10 panel a shows the plasma nitrate concentration and FIG.10 panel b shows the plasma nitrite concentration. After infusion ofnitrate the concentration of plasma nitrate increases dramatically bothin rats that received nitrate and rats that received nitrate+allopurinol(a). The plasma nitrite concentrations increased in rats that receivednitrate as well as in rats that received nitrate+allopurinol, (b).However the increase in the rats that received only nitrate wassignificantly greater, *p<0.05.

FIG. 10 panel c shows that nitrate-induced increase in plasma nitrite isequal in wild type (n=5 placebo, n=5 nitrate), germ free (n=5 placebo,n=5 nitrate) and eNOS knockout mice (n=2 placebo, n=3 nitrate), p=0.05*.

Previously it has been suggested that only bacterial cells and notmammalian cells can reduce nitrate to nitrite. These resultssurprisingly show that also mammalian cells can metabolize nitrate tonitrite. Further, they suggest that the xanthine oxidase enzyme isinvolved in the reduction of nitrate to nitrite.

6. Enhancement of Post-Ischemic Blood Flow

Rats received an intravenous bolus dose of 10 mg/kg nitrate (NaNO₃, n=4)or placebo (NaCl, n=4)) diluted in PBS (pH 7.4) followed by continuousinfusion of 3 mg/kg body weight/h. An hour after the addition of nitrate(open boxes) or placebo (filled circles), L-NAME (50 mg/kg) was given,and 10 minutes later, a supra renal clamping of the abdominal aorta wasperformed. After 30 minutes of ischemia the clamp was removed and theabdominal aortic blood flow was monitored during 60 minutes.

The results show that the nitrate-treated rats maintain a higher bloodflow during the early (0-10 minutes) as well as the late (10-60 minutes)post-ischemic phase compared to the placebo treated rats (FIG. 11).Remarkably, after 60 min of reperfusion the blood flow had decreased toonly 20% of pre-ischemic values in the control rats, while in thenitrate-treated rats, the blood flow was maintained at almost 75% ofcontrol values. This demonstrates a strong augmentation of thenitrate-nitrite-NO pathway during an ischemic event.

We claim:
 1. A method of increasing exercise endurance in a humansubject, the method comprising administering to a human subject prior toexercise a composition comprising an amount of inorganic nitrate (NO₃⁻), wherein amount of inorganic nitrate provides a supplementary amountof the inorganic nitrate.
 2. The method of claim 1, wherein the initialadministration of the composition is at least three days prior toexercise.
 3. The method of claim 1, wherein the administration of thecomposition delays the onset of fatigue during exercise.
 4. The methodof claim 1, wherein the administration of the composition reduces thesubject's oxygen consumption during exercise.
 5. The method of claim 1where the administration of inorganic nitrate increases the availableenergy to the muscle.
 6. The method of claim 1, wherein the supplementcomposition consists of the inorganic nitrate and at least one additive.7. The method of claim 6, wherein the inorganic nitrate is provided froma natural nitrate source.
 8. The method of claim 7, wherein the naturalnitrate source is juice or dried concentrate from at least one ofspinach, lettuce, fennel, cabbage, Chinese cabbage, and beetroot.
 9. Themethod of claim 6, wherein the inorganic nitrate is in the form of asalt of nitrate.
 10. The method of claim 9 wherein the salt of nitrateis selected from the group consisting of: sodium nitrate, potassiumnitrate, calcium nitrate, zinc nitrate, arginine nitrate, and nitrateammonium.
 11. The method of claim 1, wherein the supplementary amount ofthe inorganic nitrate in the composition administers between 0.01 mmolnitrate ion and 10 mmol nitrate ion per kg body weight of the subject.12. The method of claim 1, wherein the supplementary amount of theinorganic nitrate in the composition administers between 0.1 mmolnitrate ion and 1 mmol nitrate ion per kg body weight of the subject.13. The method of claim 1, wherein the supplementary amount of theinorganic acid in the composition administers 0.1 mmol nitrate ion perkg body weight of the subject.
 14. The method of claim 1, wherein thecomposition is administered to the subject once a day.
 15. The method ofclaim 1, wherein the composition further comprises a polyphenol.
 16. Themethod of claim 16, wherein the polyphenol provided from at least onenatural source selected from the group consisting of: apple, pear,grapes, lemon, orange, lime, peach, pomegranate, grapefruit, kiwi,ginger, pineapple, blackberries, black raspberries, blueberries,cranberries, red raspberries, cherries, bog wortleberry, lingonberries,black elderberry, black chokeberry, black currant, cloudberries,strawberries, carrots, chili, rhubarb, onions, cacao products, greentea, black tea, nuts, Yerba mate, and coffee.
 17. The method of claim15, wherein the composition comprises at least 50% of the salt ofnitrate.
 18. The method of claim 1, wherein the composition isadministered orally.
 19. The method of claim 1, wherein the compositionhas the form of a liquid, a paste, a bar, a cake, a powder, a granulate,an effervescent tablet, a chewing gum, a tablet, a capsule, a lozenge, afast melting tablet or wafer, a sublingual tablet or a spray.
 20. Themethod of claim 1, wherein the composition is a functional food product.21. The method of claim 20, wherein the composition is a sport drink, anenergy drink, a sport bar, or an energy bar.
 22. The method of claim 20,wherein the composition further comprises non-pathogenic live bacteriaand/or a probiotic yeast.
 23. The method of claim 22, wherein thenon-pathogenic live bacteria comprises a species selected from the groupconsisting of: Lactobacilli, Bifidobacteria, Veillonella,Staphylococcus, Actinomyces, and Rothia.
 24. The method of claim 23,wherein the Lactobacilli species is selected from the group consistingof: L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L.casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L.fermentum, and L. reuteri.
 25. The method of claim 24, wherein theBifidobacteria species is selected from the group consisting of: B.breve, B. bifidum, and B. lactis.
 26. The method of claim 15, whereinthe composition is a functional food product.
 27. The method of claim26, wherein the composition is a sport drink, an energy drink, a sportbar, or an energy bar.
 28. The method of claim 26, wherein thecomposition further comprises non-pathogenic live bacteria and/or aprobiotic yeast.
 29. The method of claim 28, wherein the non-pathogeniclive bacteria comprises a species selected from the group consisting of:Lactobacilli, Bifidobacteria, Veillonella, Staphylococcus, Actinomyces,and Rothia.
 30. The method of claim 29, wherein the Lactobacilli speciesis selected from the group consisting of: L. acidophilus, L.delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L.plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum, and L.reuteri.
 31. The method of claim 29, wherein the Bifidobacteria speciesis selected from the group consisting of: B. breve, B. bifidum, and B.lactis.
 32. The method of claim 6, wherein the additive is selected fromthe group comprising: natural flavor, artificial flavor, sweetener,flavor enhancer, color additive, emulsifier, stabilizer, fat, andpreservative.